US2021206848A1PendingUtilityA1

Receptor inhibition by phosphatase recruitment

49
Assignee: UNIV LELAND STANFORD JUNIORPriority: May 17, 2018Filed: May 16, 2019Published: Jul 8, 2021
Est. expiryMay 17, 2038(~11.8 yrs left)· nominal 20-yr term from priority
Inventors:Kenan Christopher GarciaRicardo A. Fernandes
C07K 2319/00C07K 2317/75C07K 2317/33C07K 2317/24C07K 2317/22C07K 2317/21C07K 16/2809C07K 2317/569C07K 2317/76C07K 2317/622C07K 2317/92C07K 16/2818C07K 16/289A61P 3/08C07K 2317/31A61K 2039/505A61K 45/06C12N 15/113A61P 35/00C07K 14/715A61K 38/00C07K 16/2803A61P 43/00A61P 31/12
49
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Claims

Abstract

Disclosed herein are compositions and methods for modulating cell surface receptor signaling by specifically recruiting membrane phosphatases to a proximity of receptors of interest. This novel methodology, termed Receptor Inhibition by Phosphatase Recruitment (RIPR), represents a new approach to reducing and suppressing signal by receptors that signal through phosphorylation mechanisms. More particularly, the disclosure provides novel multivalent protein-binding molecules that specifically bind a cell surface receptor and antagonize the receptor signaling through recruitment of a phosphatase activity. Also provided are compositions and methods useful for producing such molecules, as well as methods for the treatment of diseases associated with the inhibition of signal transduction mediated by cell surface receptor.

Claims

exact text as granted — not AI-modified
What is claimed is: 
     
         1 . A multivalent polypeptide comprising:
 a first amino acid sequence comprising a first polypeptide module capable of binding to one or more receptor protein-tyrosine phosphatases (RPTP); and   a second amino acid sequence comprising a second polypeptide module capable of binding to one or more cell surface receptors that signal through a phosphorylation mechanism;   wherein the first polypeptide module is operably linked to the second polypeptide module.   
     
     
         2 . The multivalent polypeptide of  claim 1 , wherein the first polypeptide module is operably linked to the second polypeptide module via a polypeptide linker sequence. 
     
     
         3 . The multivalent polypeptide of any one of  claims 1  to  2 , wherein at least one of the first and second polypeptide modules comprises an amino acid sequence for a protein-binding ligand or an antigen-binding moiety. 
     
     
         4 . The multivalent polypeptide of  claim 3 , wherein the antigen-binding moiety is selected from the group consisting of an antigen-binding fragment (Fab), a single-chain variable fragment (scFv), a nanobody, a V H  domain, a V L  domain, a single domain antibody (dAb), a V NAR  domain, and a V H H domain, a diabody, or a functional fragment of any thereof. 
     
     
         5 . The multivalent polypeptide of any one of  claims 3  to  4 , wherein the antigen-binding moiety comprises a heavy chain variable region and a light chain variable region. 
     
     
         6 . The multivalent polypeptide of  claim 3 , wherein the protein-binding ligand is a cytokine, a growth factor, a receptor extracellular domain (ECD) of a cell surface receptor or of a RPTP, or a functional variant of any thereof. 
     
     
         7 . The multivalent polypeptide of any one of  claims 1  to  6 , wherein the one or more RPTPs comprise CD45 or a functional variant thereof. 
     
     
         8 . The multivalent polypeptide of any one of  claims 1  to  7 , wherein the one or more cell surface receptors comprise an immune-checkpoint receptor, a cytokine receptor, or a growth factor receptor. 
     
     
         9 . The multivalent polypeptide of any one of  claims 1  to  8 , wherein the one or more cell surface receptors comprise an immune-checkpoint receptor selected from the group consisting of inhibitory checkpoint receptors and stimulatory checkpoint receptors. 
     
     
         10 . The multivalent polypeptide of any one of  claims 1  to  8 , wherein the one or more cell surface receptors comprises an inhibitory checkpoint receptor selected from the group consisting of PD-1, CTLA-4, A2AR, B7-H3, B7-H4, BTLA, CD5, CD132, IDO, KIR, LAG3, TIM-3, TIGIT, and VISTA or a functional variant of any thereof. 
     
     
         11 . The multivalent polypeptide of any one of  claims 1  to  8 , wherein the one or more cell surface receptors comprise a stimulatory checkpoint receptor selected from the group consisting of CD27, CD28, CD40, OX40, GITR, ICOS, and CD137 or a functional variant of any thereof. 
     
     
         12 . The multivalent polypeptide of any one of  claims 1  to  8 , wherein the one or more cell surface receptors mediate signaling through a specific tyrosine-based motif selected from an ITAM motif, an ITSM motif, an ITIM motif, or a related intracellular motif that serves as a substrate for phosphorylation. 
     
     
         13 . The multivalent polypeptide of  claim 12 , wherein the one or more cell surface receptors are selected from the group consisting of DAP10, DAP12, SIRPa, CD3, CD28, CD4, CD8, CD200, CD200R, ICOS, KIR, FcR, BCR, CD5, CD2, G6B, LIRs, CD7, and BTNs or a functional variant of any thereof. 
     
     
         14 . The multivalent polypeptide of any one of  claims 1  to  8 , wherein the one or more cell surface receptors comprise a cytokine receptor. 
     
     
         15 . The multivalent polypeptide of  claim 14 , wherein the one or more cytokine receptors are selected from the group consisting of interleukin receptors, interferon receptors, chemokine receptors, growth hormone receptors, erythropoietin receptors (EpoRs), thymic stromal lymphopoietin receptors (TSLPRs), thrombopoetin receptors (TpoRs), granulocyte macrophage colony-stimulating factor (GM-CSF) receptors, and granulocyte colony-stimulating factor (G-CSF) receptors. 
     
     
         16 . The multivalent polypeptide of any one of  claims 1  to  8 , wherein the one or more cell surface receptors comprise a growth factor receptor. 
     
     
         17 . The multivalent polypeptide of  claim 16 , wherein the growth factor receptor is a stem cell growth factor receptor (SCFR) or an epidermal growth factor receptor (EGFR) selected from the group consisting of ErbB-1, ErbB-2 (HER2), ErbB-3, ErbB-4, and c-Kit (CD117). 
     
     
         18 . The multivalent polypeptide of any one of  claims 2  to  17 , wherein the polypeptide linker sequence is about 1 to about 100 amino acid residues. 
     
     
         19 . The multivalent polypeptide of any one of  claims 2  to  18 , wherein the polypeptide linker comprises at least one glycine residue. 
     
     
         20 . The multivalent polypeptide of any one of  claims 2  to  18 , wherein the polypeptide linker comprises a glycine-serine linker. 
     
     
         21 . The multivalent polypeptide of anyone of  claims 5  to  20 , wherein the heavy chain variable region and the light chain variable region of the antigen-binding moiety are operably linked to each other via one or more intervening amino acid residues that are positioned between the heavy chain variable region and the light chain variable region. 
     
     
         22 . The multivalent polypeptide of  claim 21 , wherein the intervening amino acid residues are about 1 to about 100 amino acid residues. 
     
     
         23 . The multivalent polypeptide of any one of  claim 21  to  22 , wherein the intervening amino acid residues comprise at least one glycine residue. 
     
     
         24 . The multivalent polypeptide of any one of  claims 21  to  23 , wherein the intervening amino acid residues comprise a glycine-serine linker. 
     
     
         25 . The multivalent polypeptide of any one of  claims 1  to  24 , comprising, in the N-terminal to C-terminal direction:
 a) a domain A comprising a binding region of a heavy chain variable region of a first scFv specific for an epitope of a RPTP; 
 b) a domain B comprising a binding region of a light chain variable region of a second scFv specific for an epitope of a cell surface receptor; 
 c) a domain C comprising a binding region of a heavy chain variable region of the second scFv specific for an epitope of the cell surface receptor; and 
 d) a domain D comprising a binding region of a light chain variable region of the first scFv specific for an epitope of the RPTP. 
 
     
     
         26 . The multivalent polypeptide of any one of  claims 1  to  25 , further comprising an amino acid sequence for a signal peptide. 
     
     
         27 . The multivalent polypeptide of any one of  claims 1  to  26 , further comprising an amino acid sequence that has at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 2, 4, 6, 10, 12, 14, 16, 20, 22, 24, 26, 28, and 54. 
     
     
         28 . A multivalent antibody or functional fragment thereof comprising:
 a first polypeptide module specific for one or more receptor protein-tyrosine phosphatases (RPTPs); and   a second polypeptide module specific for one or more cell surface receptors that signal through a phosphorylation mechanism,   wherein the first polypeptide module is operably linked to the second polypeptide module.   
     
     
         29 . The multivalent antibody or functional fragment thereof of  claim 28 , wherein the first polypeptide module is operably linked to the second polypeptide module via a polypeptide linker sequence. 
     
     
         30 . The multivalent antibody or functional fragment thereof of any one of  claims 28  to  29 , wherein at least one of the first and second polypeptide modules comprises an amino acid sequence for a protein-binding ligand or an antigen-binding moiety. 
     
     
         31 . The multivalent antibody or functional fragment thereof of  claim 30 , wherein the antigen-binding moiety is selected from the group consisting of an antigen-binding fragment (Fab), a single-chain variable fragment (scFv), a nanobody, a V H  domain, a V L  domain, a single domain antibody (sdAb), a V NAR  domain, and a V H H domain, or a functional fragment thereof. 
     
     
         32 . The multivalent antibody or functional fragment thereof of any one of  claims 30  to  31 , wherein the antigen-binding moiety comprises a heavy chain variable region and a light chain variable region. 
     
     
         33 . The multivalent polypeptide of  claim 30 , wherein the protein-binding ligand is a cytokine, a growth factor, a receptor extracellular domain (ECD) of cell surface receptor, or a functional variant of any thereof. 
     
     
         34 . The multivalent antibody or functional fragment thereof of any one of  claims 28  to  33 , wherein the one or more RPTPs comprise CD45 or a functional variant thereof. 
     
     
         35 . The multivalent antibody or functional fragment thereof of anyone of  claims 28  to  34 , wherein the one or more cell surface receptors comprise an immune-checkpoint receptor, a cytokine receptor, or a growth factor receptor. 
     
     
         36 . The multivalent antibody or functional fragment thereof of any one of  claims 28  to  35 , wherein the one or more cell surface receptors comprise an immune-checkpoint receptor selected from the group consisting of inhibitory checkpoint receptors and stimulatory checkpoint receptors. 
     
     
         37 . The multivalent antibody or functional fragment thereof of any one of  claims 28  to  36 , wherein the one or more cell surface receptors comprise an inhibitory checkpoint receptor selected from the group consisting of PD-1, CTLA-4, A2AR, B7-H3, B7-H4, BTLA, CD5, CD132, IDO, KIR, LAG3, TIM-3, TIGIT, and VISTA or a functional variant of any thereof. 
     
     
         38 . The multivalent antibody or functional fragment thereof of anyone of  claims 28  to  37 , wherein the one or more cell surface receptors comprise a stimulatory checkpoint receptor selected from the group consisting of CD27, CD28, CD40, OX40, GITR, ICOS, and CD137 or a functional variant of any thereof. 
     
     
         39 . The multivalent antibody or functional fragment thereof of any one of  claims 28  to  35 , wherein the one or more cell surface receptors mediate signaling through a specific tyrosine-based motif selected from an ITAM motif, an ITSM motif, an ITIM motif, or a related intracellular motif that serves as a substrate for phosphorylation. 
     
     
         40 . The multivalent antibody or functional fragment thereof of  claim 39 , wherein the one or more cell surface receptors are selected from the group consisting of DAP10, DAP12, SIRPa, CD3, CD28, CD4, CD8, CD200, CD200R, ICOS, KIR, FcR, BCR, CD5, CD2, G6B, LIRs, CD7, and BTNs or a functional variant of any thereof. 
     
     
         41 . The multivalent antibody or functional fragment thereof of anyone of  claims 28  to  35 , wherein the one or more cell surface receptors comprise a cytokine receptor. 
     
     
         42 . The multivalent antibody or functional fragment thereof of  claim 41 , wherein the one or more cytokine receptors is selected from the group consisting of interleukin receptors, interferon receptors, chemokine receptors, growth hormone receptors, erythropoietin receptors (EpoRs), thymic stromal lymphopoietin receptors (TSLPRs), thrombopoetin receptors (TpoRs), granulocyte macrophage colony-stimulating factor (GM-CSF) receptors, and granulocyte colony-stimulating factor (G-CSF) receptors. 
     
     
         43 . The multivalent antibody or functional fragment thereof of any one of  claims 28  to  35 , wherein the one or more cell surface receptors comprise a growth factor receptor. 
     
     
         44 . The multivalent antibody or functional fragment thereof of  claim 43 , wherein the growth factor receptor is a stem cell growth factor receptor (SCFR) or an epidermal growth factor receptor (EGFR) selected from the group consisting of ErbB-1, ErbB-2 (HER2), ErbB-3, ErbB-4, and c-Kit (CD117). 
     
     
         45 . The multivalent antibody or functional fragment thereof of any one of  claims 28  to  44 , wherein the polypeptide linker sequence comprises 1-100 amino acid residues. 
     
     
         46 . The multivalent antibody or functional fragment thereof of  claims 29  to  45 , wherein the polypeptide linker comprises at least one glycine residue. 
     
     
         47 . The multivalent antibody or functional fragment thereof of  claims 29  to  46 , wherein the polypeptide linker comprises a glycine-serine linker. 
     
     
         48 . The multivalent antibody or functional fragment thereof of  claims 32  to  47 , wherein the heavy chain variable region and the light chain variable region of the antigen-binding moiety are operably linked to each other via one or more intervening amino acid residues that are positioned between the heavy chain variable region and the light chain variable region. 
     
     
         49 . The multivalent antibody or functional fragment thereof of  claim 48 , wherein the intervening amino acid residues are about 1 to about 100 amino acid residues. 
     
     
         50 . The multivalent antibody or functional fragment thereof of any one of  claims 48  to  49 , wherein the intervening amino acid residues comprise at least one glycine residue. 
     
     
         51 . The multivalent antibody or functional fragment thereof of anyone of  claims 48  to  50 , wherein the intervening amino acid residues comprise a glycine-serine linker. 
     
     
         52 . The multivalent antibody or functional fragment thereof of  claims 28  to  51 , comprising, in the N-terminal to C-terminal direction:
 a) a domain A comprising a binding region of a heavy chain variable region of a first scFv specific for an epitope of a RPTP; 
 b) a domain B comprising a binding region of a light chain variable region of a second scFv specific for an epitope of a cell surface receptor; 
 c) a domain C comprising a binding region of a heavy chain variable region of the second scFv specific for an epitope of the cell surface receptor; and 
 d) a domain D comprising a binding region of a light chain variable region of the first scFv specific for an epitope of the RPTP. 
 
     
     
         53 . The multivalent antibody or functional fragment thereof of any one of  claims 28  to  52 , further comprising an amino acid sequence for a signal peptide. 
     
     
         54 . The multivalent antibody or functional fragment thereof of any one of  claims 28  to  53 , comprising an amino acid sequence that has at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOS: 2, 4, 6, 10, 12, 14, 16, 20, 22, 24, 26, 28, and 54. 
     
     
         55 . A pharmaceutical composition comprising:
 a multivalent polypeptide according to any one of  claims 1 - 27 , or   a multivalent antibody or a functional fragment thereof according to any one of  claims 28 - 54 ,   and a pharmaceutical acceptable excipient.   
     
     
         56 . A recombinant nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide that comprises:
 a) an amino acid sequence having at least 80% identity to the amino acid sequence of the multivalent polypeptide of any one of  claims 1 - 27 ; or   b) an amino acid sequence having at least 80% identity to the multivalent antibody of or a functional fragment thereof according to any one of  claims 28 - 54 .   
     
     
         57 . The recombinant nucleic acid molecule of  claim 56 , wherein the nucleotide sequence has at least 80% sequence identity to a nucleotide sequence selected from the group consisting of SEQ ID NOS: 1, 3, 5, 9, 11, 13, 15, 19, 21, 23, 25, 27, and 53. 
     
     
         58 . An expression cassette or a vector comprising the recombinant nucleic acid molecule of any one of  claims 56 - 57 . 
     
     
         59 . A recombinant cell comprising the recombinant nucleic acid molecule of any one of  claims 56 - 57 . 
     
     
         60 . A cell culture comprising one or more recombinant cells of  claim 59  and a culture medium. 
     
     
         61 . A method for producing a polypeptide or a multivalent antibody comprising:
 providing one or more recombinant cells of  claim 59 ; and   culturing the one or more recombinant cells in a culture medium such that the cells produce the multivalent polypeptide or the multivalent antibody encoded by the recombinant nucleic acid molecule.   
     
     
         62 . A method for modulating cell signaling mediated by a cell surface receptor that signals through a phosphorylation mechanism in a subject, the method comprising administering to the subject a first therapy comprising an effective amount of
 a) a multivalent polypeptide according to any one of  claims 1 - 27 ; or   b) a multivalent antibody or a functional fragment thereof according to any one of  claims 28 - 54 .   
     
     
         63 . A method for the treatment of a disease in a subject in need thereof, the method comprising administering to the subject a first therapy comprising an effective amount of
 a) a multivalent polypeptide according to any one of  claims 1 - 27 ; or   b) a multivalent antibody or a functional fragment thereof according to any one of  claims 28 - 54 .   
     
     
         64 . The method of any one of  claims 62  to  63 , wherein the administered multivalent polypeptide or the multivalent antibody recruits the receptor protein-tyrosine phosphatase (RPTP) activity to a spatial proximity of the cell surface receptor and reduces phosphorylation level of the cell surface receptor. 
     
     
         65 . The method of any one of  claims 62  to  64 , wherein the administration of the multivalent polypeptide or the multivalent antibody confers reduced activity of an immune checkpoint receptor in the subject. 
     
     
         66 . The method of any one of  claims 62  to  64 , wherein the administration of the multivalent polypeptide or the multivalent antibody confers an enhancement in T-cell activity in the subject. 
     
     
         67 . The method of any one of  claims 62  to  64 , wherein the administration of the multivalent polypeptide or the multivalent antibody confers suppression of T-cell activity in the subject. 
     
     
         68 . The method of any one of  claims 62  to  67 , wherein the subject is a mammal. 
     
     
         69 . The method of  claim 68 , wherein the mammal is a human. 
     
     
         70 . The method of any one of  claims 62  to  69 , wherein the subject has or is suspected of having a disease associated with inhibition of cell signaling mediated by the cell surface receptor. 
     
     
         71 . The method of  claim 70 , wherein the disease is a cancer or a chronic infection. 
     
     
         72 . The method of anyone of  claims 62  to  71 , further comprising administering to the subject a second therapy. 
     
     
         73 . The method of  claim 72 , wherein the second therapy is selected from the group consisting of chemotherapy, radiotherapy, immunotherapy, hormonal therapy, or toxin therapy. 
     
     
         74 . The method of any one of  claims 72  to  73 , wherein the first therapy and the second therapy are administered concomitantly. 
     
     
         75 . The method of any one of  claims 72  to  74 , wherein the first therapy is administered at the same time as the second therapy. 
     
     
         76 . The method of any one of  claims 72  to  73 , wherein the first therapy and the second therapy are administered sequentially. 
     
     
         77 . The method of  claim 76 , wherein the first therapy is administered before the second therapy. 
     
     
         78 . The method of  claim 76 , wherein the first therapy is administered after the second therapy. 
     
     
         79 . The method of any one of  claims 72  to  73 , wherein the first therapy is administered before and/or after the second therapy. 
     
     
         80 . The method of any one of  claims 72  to  73 , wherein the first therapy and the second therapy are administered in rotation. 
     
     
         81 . The method of anyone of  claims 72  to  73 , wherein the first therapy and the second therapy are administered together in a single formulation

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